Infrared spectroscopy of the structure-sensitive amide I band of proteins will be applied to the study of guanidine-HCl-induced protein denaturation of a broad range of proteins. Particular emphasis will be placed on analysis of structure in the denatured state. Given the increased interest in the possible role of the denatured states of proteins in their folding properties such data will be of wide interest. Guanidine-HCl denaturation is one of the most used methods to study protein thermodynamics. Structural information on the guanidine-HCl-denatured state is therefore very important. The pharmaceutical industry is also currently very interested in the effects of protein denaturation in drug formulations. Improved spectroscopic methods and understanding of the denatured state of proteins could be crucial to this industry. Infrared spectroscopy has a number of advantages for studying protein denaturation. It is fast, inexpensive and not subject to conformational averaging effects as with NMR spectroscopy. Proteins will be analyzed using the infrared technique for monitoring guanidine-HCl denaturation previously developed in our laboratory using iso-1-cytochrome c. The proteins of choice, 14 lysozyme, RNase A, RNase T1, apomyoglobin and staphylococcal nuclease are all widely studied models for protein folding and cover a range of native state structural types. Spectra will be acquired by Fourier transform infrared methods in H2O solution using 6 mu m pathlength cells. Buffer and water vapor will be subtracted, followed by careful subtraction of the guanidine-HCl infrared band. After Savistsky-Golay smoothing the spectra will be deconvoluted using second derivative methods. Assignment of structural types will be based on existing empirical assignments of the amide I band. To enhance the sequence-specific resolution of this method, preparation of 13C-labelled proteins using semisynthesis methods is also proposed. This work will involve mutagenesis of iso-1-cytochrome c to produce cyanogen bromide cleavage sites in front of the C-terminal alpha-helix. Using the well-studied methods of cyanogen bromide cleavage at methionine residues followed by autocatalytic fragment relegation, 13C-labelled carbonyls will be introduced at specific positions in the C-terminal alpha-helix. The 37 cm(-1) shift in the amide I band produced by this labelling will allow direct assignment of the inked band resulting from the C-terminal alpha- helix. This assignment will be important in clarifying existing ambiguities in secondary structure assignment of the amide I band. The labelled proteins will also allow the effects of guanidine-HCl denaturation on a specific segment of iso-1-cytochrome c to be observed directly.